The central hypothesis of this program project is that down-regulation of presynaptic opioid receptors on primary afferent fibers leads to disinhibition of excitatory neurotransmitter release and apparent tolerance to the analgesic effects of opioids. Other investigators in this project will correlate receptor down-regulation with analgesic tolerance in vivo, and with regulation of evoked neurotransmitter release in vitro. We propose to study the basic cellular mechanisms of human 5-opioid receptor (hDOR) down-regulation in a recombinant model system (hDOR/CHO cells). We recently made the novel observations that a. down-regulation of the hDOR in CHO cells is sensitive to proteasomal, but not to lysosomal inhibitors; b. deletion of the cytoplasmic tail of the hDOR (trunchDOR) blocks down-regulation by DPDPE, but not by SNCS0; c. SNC 80-mediated residual downregulation of the truncated hDOR is sensitive to lysosomal but not to proteasomal inhibitors. Based on these findings, we hypothesize that structurally distinct opioid agonists mediate opioid receptor down-regulation through different mechanisms. In Specific aim I of this proposal we intend to confirm the preliminary findings by using more specific methods (dominant negative mutants) to inhibit lysosomal and proteasomal pathways in hDOR/CHO and trunc-hDOR/CHO cells. In Specific aim II we will determine the role of proteasomal targeting motifs in the C-terminus of the hDOR in DPDPE, SNC 80 and biphalin-mediated receptor down-regulation. In Specific aim III we will study the role of lysosomal targeting motifs in SNC 80 mediated residual down-regulation of the truncated hDOR. Better understanding of the mechanisms of hDOR down-regulation during chronic agonist treatment may lead to development of opioid analgesics that produce limited analgesic tolerance.